Phthalide penicillin ester and salts

ABSTRACT

Phthalide ester of 6-(D(-) Alpha -aminophenylacetamido) penicillanic acid, intermediates therefor, process for preparation and pharmaceutically acceptable acid addition salts are described. The phthalide penicillin ester is characterized by high serum concentrations of the parent penicillin upon oral administration.

United States Patent [1 Ferres et al.

[ Jan. 14, 1975 EHTHALIDE PENlClLLlN ESTER AND SALTS [75] Inventors:Harry Ferres; John Peter Clayton,

both of Horsham, England [73] Assignee: Beecham Group Limited,Brentford,

Middlesex, England [22] Filed: June 5, 1972 [21] Appl. No.: 259,560

[30] Foreign Application Priority Data June 9, 1971 Great Britain19604/71 [52] [1.5. Cl. 260/2311, 424/271 [51] Int. Cl C07d 99/22 [58]Field of Search 260/239.1

[56] References Cited UNITED STATES PATENTS 3,697,507 10/1972Frederiksen et ul 260/2391 Primary Examiner-Nicholas S. Rizzo 2 Claims,N0 Drawings PHTHALIDE- PENICILLIN ESTER AND SALTS This invention relatesto the pathalide ester of 6- [D(-)a-aminophenylacetarnido] penicillanicacidv and its pharmaceutically acceptable acid addition salts, and to aprocess'for its preparation.

6-[D(-)a-aminophenylacetamido] 'penicillanic acid is a widely used broadspectrum antibiotic. However,

' when administered orally, it is incompletely absorbed into thebloodstream. Some medical practitioners believe this to be adisadvantage and consequently some attempts have been made to findderivatives of 6-[I)(- )waminophenylacetamidol penicillanic acid whichwill produce higher blood concentrations of the parent pen- The preterred acid addition salt of the compound of this invention is thehydrochloride, but salts with other 2 eral halotrialkylsilanes arepreferred, especially trimethylchlorosilane. The silylated I derivativesof 6- aminopenicillanic acid phthalide ester areextrernely sensitive tomoisture and hydroxylic compounds,.-and

after reaction withthe reactive derivative of compound (II), the silylgroup of the intermediate acylated compound can be removed by hydrolysisor alcoholysis. In compound (II) the group X is anamino group, protectedamino group which. is convertible to an amino group.

Examples of protected amino groupinclude the pro tonated amino groups (XNH?) which, after the acylation reaction can be eommited to a free aminogroup by simple neutralisation; the benzyloxycarbonylamino group (XNH.CO CH Ph) or substituted benzyloxycarbonylamino groups whicharesubsequently 'converted to NH by catalytic hydrogenation; and variousgroups which after the acylation reaction regenerate the amino group onmild acid hydrolysis. (Alkalinehydrolysis is not generally useful sincehydrolysis of the phthalide grouptakes place under alkaline conditions).

Examples of the group X which may subsequently be converted to NH bymild acid hydrolysis include 'enamine groups of general formula (III) ortautomeric modifications thereof, and a-hydroxyarylidene groups ofgeneral formula (IV) or tautomeric modifications thereof:

inorganic or organic acids may be used (especially those acids whichhave been employed to form salts with 6-[D(-)a-aminophenylacetamido]-penicillanic' acid itself). In addition tothe .compound'of thisinvention will form salts with other penicillanic-acids e.g.,3-'(2-chloro 6-fluorophenyl) 5-methyl-4- isoxazolyIpe-nicillin.

The ester of the present invention may be prepared by a process 1 whichcomprises reacting I 6- aminopenicillanie acid phthalide ester or asilyl deriva- -tive thereof with a reactive N-acylating derivative ofthe (D) isomer of a compound of formula (II):

Goa-0001a wherein X is an amino group, a protected amino group or agroup which is convertible to an amino group, removing the silyl group,if present, by hydrolysis or alcoholysis, and if X is not an aminogro'up,'.converting it to such a group under neutral or acid conditions.

By the term silyl derivative" of the phthalide ester ofo-aminopenieillanic acid we mean the product of the reaction betweeno-aminopenicillanic acid phthalide ester and a silylating agent such asa halotrialkylsilane; a dihalodialkylsilane, a halotrialkylsilane, adihalodialkoxysilane or a corresponding aryl or aralkyl silane andcompounds such as -hexarnethyldisilazane. In gen- In structures (III)and (IV), the dotted lines represent 7 hydrogen-bonds. In structure(III) R is a lower alkyl group, R is either a hydrogen atom or togetherwith R completes a carbocyelic ring, and R isa lower alkyl, aryl, orlower alkoxy group. In structure (IV) X rep-resents the residue of asubstituted or unsubstituted benzene or naphthalene ring. I

An example of a group X which can be converted to NH -after theacylation reaction of the phthalide ester of 6-aminopenicillanic acidwith the reactive derivative of (II) is the azido group. In this case,the final conversion into NI-I maybe brought about by either catalytichydrogenation or electrolytic reduction.

A reactive N-acylating derivative of the acid (II) is employed in theabove process. The choice of reactive derivative will of coursebeinfluenced by the chemical nature of the a-substituent X. Thus, when Xis an acid stable group, such as the protonated amino group NI-I or theazido group, it is often convenient to convert the acid (II) into anacid halide, for example by treating it with thionyl chloride orphosphorus'pentachloride' to give the acid chloride.

Such reagents would however be avoided when X is an acid labile group oftype (III) or (IV). In such cases it is often convenient to make use ofa mixed anhydride. For this purpose particularly convenient mixedanhydrides are the alkoxyformic anhydrides, which are convenientlyprepared by treating an alkali metal or tertiary amine salt of the acid(II) with the appropriate alkyl chloroformate in an anhydrous medium ator below room temperature.

Other reactive N-acylating derivatives of the acid (II) include thereactive intermediate formed on reaction in 'situ with a carbodiimide orcarbonyldiimidazole.

The o-aminopenicillanic-acid phthalide ester used in the above processcan be prepared, though in poor yield by direct couplingof'o-amino-penicillanic acid with 3-bromophthalide in the presence of abase. With this process some epimerisation of C occurs and the processis therefore not entirely satisfactory.

o-Aminopenicillanic acid phthalide ester is also a new compound, and,since it is a valuable intermediate in the process of this invention, italso forms part of the invention. Much better yields of-aminopenicillanic acid phthalide ester can be achieved by coupling anN- protected derivative of 6-aminopenicillanic acid (e.g. thetriphenylmethyl derivative) with 3-bromophthalide and thereafterremoving the protecting group (e.g. by mild acid hydrolysis in the caseof the triphenylmethyl derivative).

Alternativetypes of N-protected -6-amino penicillanic acid are the6-acylaminopenicillanic acids. Techniques for the removal of the 6-acylside chain from benzylpenicillin and phenoxymethyl penicillin, forexample, are well documented (of. British Pat. No. 1,189,022) andgenerally involve treating an ester of the -acylaminopenicillanic acidwith PCI to form an imino chloride bond on the 6-amido nitrogen atom,then treating the imino chloride with an alcohol to form an imino etherand then hydrolysing the imino bond to 'form the o-aminopenicillanicacid ester. In the present the phthalido group.

Thus the invention provides in another of its aspects, a method for. thepreparation of 6[D(-)aaminophenylacetamido] penicillanic acid phthalideester of formula (I) above, which process comprises reacting a compoundof formula (V):

CII:

or a reactive esterifying derivative thereof, in which formula X is asdefined with respect to formula (II), with a compound of formula (VI):

HO\C H or a reactive esterifying' derivative thereof, and, if X is notan amino group, subsequently converting it to an amino group underneutral or acid conditions.

By the term reactive esterifying derivative" in relation to compounds(V) and (VI) above, we mean deravatives of (V) and (VI) which whenreacted together take part in a condensation reaction with theconsequent formation of anester linkage:

COO II wherein X is as defined with respect to formula (II) above, witha compound of formula (WA):

(VIA) under conditions which cause the elimination of the elements ofcompound AB with the consequent formation of the ester of formula (VII):

(VII) and, if X is not an amino group, subsequently converting it to anamino group; the symbols A and B in formulae (VA) and (VI) being suchthat A represents hydro gen or a salt forming ion and B represents ahydroxy group,

an alkylsulphonyloxy group, an arylsulphonyloxy group or a halogen atomor A represents an organic acyl group and B represents a hydroxy group.

It will be clear that these procedures outlined above are all specificapplications of esterification methods known in the literature. Althoughthe group X in reagent (V) may be a free amino group, the reaction isbest carried out with reagent (V) where X is a protcctcd amino group(preferably an enaminc group of formula (111)) or a group which can beconverted to an amino group (eg. azido). In such cases it is usually asimple matter to react the sodium or potassium salt of the N-protectedcompound (V) with compound (Vl) wherein B is a halogen atom, especiallybromine or chlorine.

1n the case of the reaction where the group A in reagent (V) ishydrogen'or a saltforming ion and the group B in reagent (V1) is ahydroxy group, it should be noted that the hydroxy compound (VlB) is infact in equilibrium as follows:

(VIB) ally necessary to achieve high yields.

. The following Examples illustrate the invention:

EXAMPLE 1 v a. 3-Bromophthalide l3-Bromo-l -(3H)' lsobenzofuranone]Phthalidc (10.0 g; 0.075 moles) and Nbromosuccinimide were refluxed indry carbon tetrachloride (200 ml.) in the presence of a catalytic amountof a-a2o-iso-' butyronitrile for 34 hours. The end ofthc reaction wasindicated by the disappearance of N-bromosuccinimide from the bottom ofthe reaction vessel and the accumulation of succinimide at the top. Thesuccinimide was removed by filtration and the filtrate concentrated invacuo to l5-20 rnl. Cooling of this concentrate'followed by filtrationgave 13.0 g. (81 percent yield) of crude 3-bromophthalide, m.p. 75-80,as a white crystalline solid. The product was recrystallised fromcyclohexane as colourless plates, in m.p. 78-80 with a 95 percentrecovery. N.m.r. (CCl 8= 7.67 (4H.m. aromatic), 8 7.38 (1H.s. CH) b.D(-)a-aminobenzylpenicillinphthalide ester, hydrochloride AnhydrousD(-)a-aminobenzylpenicillin (17.5; 0.05

mole) and triethylamine (7.10 ml; 1 equiv.) were.

mixed with acetone ccontaining 1 percent of water (350 ml). After onehalf hour potassium bicarbonate (5 In the case where the group A inreagent (V) is an or- I ganic acyl group, it will be clear that (V) issimply a mixed anhydride, the acyl group may be one of a wide variety ofaliphatic or aromatic acyl groups but generally the alkoxy carbonylgroups (eg. C 1-1 OCO- group) are satisfactory. I

Another reactive esterifying derivative of compound (V) above is theacid halide, particularly the acid chloride. The compound may be reactedwith the hydroxy compound (V1) in the presence of an acid binding agentto prepare the desired phthalide ester of this invention.

The identity .of the various protected amino groups X in formula (V)have already been discussed earlier in this specification with referenceto formula (11).

The compound of this invention is well tolerated and is preferablyadministered orally, optionally in the form of an acid addition salt.Usually it will be administered in combination with suitablepharmaceutically acceptable carriers. In such compositions the compoundof this invention may make up between 1 percent and 95 percent by weightof the total composition. The composition may be presented as a powderfor making up into a syrup, as a tablet, capsule or pill or any otherconvention form. v

The ester of this invention, or its salts, may conveniently beadministered in dosage units containing the equivalent of from 0.025 gto l g of 6 [D(-)ozaminophenylacetamido] penicillanic acid, preferablythe equivalent of from 0.1 to 0.7 g of the penicillanic acid. Dosageunits containing the equivalent of 250 mg or 500 mg of the parentpenicillanic acid may be found convenient. Daily dosages will depend onthe condition of the patient but generally from 1 to 3 g of the ester ofthis invention (calculated as the parent penicillanic acid) will usuallybe appropriate.

The compound of this invention 6[D(-)aaminophenylacetamido] penicillanicacid phthalide ester is well absorbed when given to human beings andanimals by the oral route. In the serum, high levels of the parent6[D(-)a-aminophenylacetamido] penicillanic acid are achieved.

g) and 3-bromophthalide (10.65 g; 0.05 mole) were added and the mixturestirred at room temperature for 4 hours. After filtration, the filtratewas concentrated in vacuo to about ml. ethyl acetate (500 ml) was addedand the resulting solution washed with a 2 percent aqueous solution ofsodium bicarbonate (2 X ml) followed by water (2 X 100ml). Water ml) wasadded to the ethyl acetate solution, and, with vigorous stirring, 1Nhydrochloric acid was added drop by drop until the pH of the aqueousphase was 2.5. The ethyl acetate layer was separated and dried overanhydrous magnesium sulphate. Ether was then added to the clear yellowethyl acetate filtrate until no further precipitation ofa whiteamorphous solid occurred. The product was collected (7.8 g; 28.8percent). Further material (0.8 g; 3.0 percent), was obtained from theaqueous layer as follows. To the aqueous layer, nbutanol (750 ml) wasadded and the resulting mixture was evaporated in vacuo until all thewater was removed. The resulting butanolic solution was poured intoether (2000 ml) whereby an amorphous precipitate separated. Combinedyields were 31.8 percent. The IR. spectrum (KBr) contains inter aliastrong bonds at:

1778 cm 1149 cm 1682 cm- 978 cm 1500 cm 752 cm 1285 cm 697 cm 5.16(111.5. a-proton); 8= 4.54 (ll-Ls. C proton); .6

1.45 (6H.d. gem-dimethyls). The purity as assessed by hydroxylamine andcysteine assays was 92.4 percent and 86.5 percent respectively. C H O NSCI requires:

C, 55.65 N, 4.67; N, 8.11; S, 6.19; Cl, 6.84. Found: C,

54.49; H, 4.67; N, 7.83; S, 6.20; Cl, 5.18.

EXAMPLE 2 Phthalide 6-[D(-)a-aminophenylacetamido1 penicillanate,Hydrochloride I Method 1 A fine suspension of potassium salt of enamineprotectd ampicillin I (25.18 g; 0.05 M) and 3- bromophthalide (10.65 g;0.05 M) were reacted in a 1:2 mixture of acetone/ethyl acetate. (1500ml)for 24 hours. After filtration the organic layer was washed.

twice with 250 ml. portions of IN sodium bicarbonate and brine, driedover anhydrous magnesium sulphate and concentrated in vacuo. Addition ofether crystallised the phthalide enamine protecteda-am'inophenylacetamido penicillanate (11) in 85 percent yield. n.m.r.[(CD SO] :8 7.86 (4l-l.m. phthalide aromatics); 8 7.60 (ll-Ls. C0.0.CH);8 7.35 (SI-Ls. aromatics) 5 5.30 5.65'(3H.m B lactams and a-proton; 84.53 (1H.s. C-3 proton) 5 4.50

(1H.s. Mg)

6 3.56 (3H5. CH3) 1.78

(3H.s. CH3) 8= 1.50 (6H.m.gemadiCl-1 ).C H N O S requires: C, 59.26; H,5.11; N, 7.40 S, 5.68. Found: C, 58.8; H, 5.00;1N, 6.89 S, 5.34. Singlespot on biochromatogram at R The enamine protecting group was removedfromthe product (II) by dissolving 10 g. in-aqueous acetone (250 mlwater to 250 ml acetone) and vigorously stirring this solution at pH 2.5for 1 hour. The'acetone was removed in vacuo and the ester (III), whichwas salted out of the aqueous phase as a sticky yellow gum, wasdissolved in ethyl acetate (200 ml) and washed twice with 200 ml portionof 1N sodium bicarbonate and brine and dried over anhydrous magnesiumsulphate. Careful addition ofdry ester (ca.50 ml) to the dry ethylacetate layer yielded the ampicillin phthalide ester as hydrochloricsalt as a fine white amorphous solid in 80 percent yield. n.m.r. [(CDSO/D O); 8 7.88

' sample (4H.m. phthalide aromatics);'6 7.60 ('1H.s. C0.0 CH-); 8 7.48(5/6H.m. aromatic) 8m 5.50 (2H.m. ,B-lactams); 8 5.16 (Tl-Ls. a-proton)6 4.54 (1H.s. C proton) 8 1.45 (6H.d. gem-dimethyls). Purity as assessedby hydroxylamine assay 1 10.3 percent. Single spot on biochromatogram atR 0.85 C24H24N305SC1 requires: C, N, S, 6.19. Found C, 54.60 H, 4.70; N,7.92; S, 6.40 METHOD 2 A mixture of acetone (250)ml, sodium D(+)N-methoxy carbonylpropen-Q-Yl-a-aminophenylucetate (30.5 g), ethylchloroformate (109ml) and N-methyl morpholine (4-6 drops) were stirredtogether for 10-15 minutes at 20 to 30C.

. To this solution was added, all at once a solution of 6-APA (25.4 g)dissolved in water (50ml) with the aid of triethylamine (1 1.9 g) andthen diluted with acetone (150 ml) and cooled to 20C. 1

The reaction mixture was stirred for 45 mins. without further coolingand a solution of 3-bromophthalide (25 g) in acetone ml) added all atonce, after which stirring was continued for a further 5 hours, thetemperaturerising meanwhile to ambient (23C.).

Acetone was next removed in vacuo, after first clarifying the mixture byfiltration and to the residue was added ethyl acetate (375 ml.) and 2percent sodium bicarbonate solution (200ml.). After stirring for a shortwhile the phases were separated and the organic layer washed again with2 percent sodium bicarbonate solution (200 ml.).

To the ethyl acetate solution thus obtained was added water (375 ml) and2N/HC1 (60ml) and charcoal. this mixture stirred at ambient. temperature(23C) for 45 mins. Petrol (600 ml) was then added and after a shortperiod of stirring the phases were allowed to separate. The organiclayer was discarded and the aqueous layer was filtered with a littledeclourising decolourising -aminophenylacetamido)] Sufficientsodium'chloride to saturate the filtrate was next added and after a fewminutes stirring the precipitated oil was extracted with methylenedichloride 1 X 400 ml, 1 X 100 ml). These extracts were combined, driedwith anhydrous magnesium sulphate,.filtered and evaporated under reducedpressure to approximately 100 m1. Ether (500ml) was then added quickly,with stirring to the residue and the resulting precipitate stirred forabout 30 mins. at ambient temperature. The product was filtered at thepump, washed with ether (2 X '50 ml) and dried for 3 hours in aforced-air oven at 35 40C. The product was identical with an authenic ofphthalide 6-[D(-)aaminophenylacetamido)] penicillanate.

EXAMPLE 3 Phthalide -aminopenicillanate. Method 1 A mixture of6-aminopenicillanic acid (10.8 g 0.05 M) and triethylamine (6.9 ml; 0.05M) was stirred in dry acetone (20 ml) for one-half hour at ambienttemperatures. The mixture was cooled to 0C and a solution of3-bromophthalide (10.65 g 0.05 M) in dry acetone (20 ml) added in oneportion and the resulting yellow mixture stirred at ambient temperaturesfor 5 hours. The reaction mixture was diluted with dry diethyl ether ml)and filtered. The clear yellow filtrate was washed with 1N sodiumbicarbonate (100 ml) and a saturated brine solution (100 ml). A solutionof -toluenesulphonic acid monohydrate (9.5 g- 1 equiv.)

in dry acetone (150 ml) was added to the clear dry yeln.m.r. [(CD SO] :L8 7.84 (4H.s. phthalide aromatics)- 8 7.58 (1H.s. CO. O.CH); 8 7.30(4H.q. sulphonate aromatics); 8 5.35 (1H.d. C 5 proton J 2H2); 8 4.89(1H.s. C 3 proton); 8 4.70 (1H.d. C- 6 proton- J 2H2) 8 2.30 (3H.s.CH3); 8 1.48 (6H.d. gemi-di CH i.r. (KBr disc) strong bands at 1780cm,1210 cm"; 1170 cm; 1010 cm; 970 cmm 682 cm; 574 cm.

A residual oil was obtained from the mother liquor from which a smallamount (ca. 5 percent of a reasonably pure sample of the natural cisisomer of'phthalide 6-aminopenicillanate asits ptoluenesulphonate saltwas obtained by repeated fractional crystallisation of the 6-a(trans)isomer from an acetonezether (3:1) solvent mixture.

6-B(cis) isomer I n.m.r. [(CD SO]: 8= 7.84 (4H.s. phthalidearomatics) 87.58 (1H.s. CO CH); 8 7.30 (4H.q. sulphate aromatics); 8 5.50 (1H.d. C 5proton; J

4H2) 8 5.14 (1H.d. C- 6 proton) J 4H2) 8 4.68

(1H.s. C 3 proton) 8 2.27d(3H.s. CH 8 1.53 (6H.d. gemdimethyls). i.r.(KBr disc) strong bands at 1780 cm" ;12l0 cm; 1170 cm" 1010 cm 970 cm"682cm 574 cm.

Method 2 A solution of 6-tritylaminopenicillanic acid (9.8 g;'

0.02M) in dry acetone (100 ml) was cooled to 0C, triethylamine (2.9 mg,0.02 M) was added, followed by 3-bromphthalide (4.1 g; 0.02M) in dryacetone ml.), and the reaction mixture kept at 0C with stirring for 2hours and finally at ambient temperatures for 1 hour. Thetriethylammonium bromide which precipitatecl was removed by filtration,the evaporatedfiltrate was dissolved in ethyl acetate (150 ml) and aftertwo washings with cold 2 percent aqueous sodium bicarbonate (2 X 150 ml)and with icewater (2 X 100 ml) the ethyl'acetate layer was dried overanhydrous magnesium sulphate to yield, on removal of solvent in vacuo,phthalide 6-tritylaminopenicillanate as a white amorphous solid. n.m.r.[(CD SO] 8 7.4 (20 H. broad singlet with smaller shoulderat 8 =7.80aromatic protons and CO 0 CH) S 4.41 (2H.m. B-lactam protons); 8 4.15(1H broad singlet C-3 proton); 8 1.38 (6H.d. gem dimethyls). i.r.(KBrdisc) strong bands at 1745 cm"; 980 cm 750 cm 708 cm.

Phthalide 6-tritylaminopenicillanate (5.9 g; 0.01 m) in acetone (200 mlcontaining 0.2 percent H O) was treated with p-toluenesulphonic acidmonohydrate (1.9 g; 0.01 'M). After standing at room temperature for 2hours, water (025 ml) was added, and the precipitation of phthalide6-aminopenicillanate p-toluenesulphonate was achieved by the slowaddition of petroleum ether, b.p. 4060 (250 ml). Filtration andconsecutive washings with petroleum ether left the crudep-toluenesulphonate salt. The sample was recrystallised fromacetone-diethyl ether with an 85 percent recovery. n.m.r. [(CD SO]: 87.84 (4H.s. phthalide aromatics); 8 7.58 (1H.s. C0.0 CH); 8 7.30 (44.9q. sulphonate aromatics)- B 5.50 (1H.d. B-lactams; J 4H2); 8 5.14 (1H.d.B -lactam, J 4H2); 8 4.68 (1H.s. C 3 proton); 8 2.27 (3H.s. CH 8 1.53(6H.d. gem dimethyls). C H N S O requires: C,

53.08; H, 4.61; N, 5.39; S, 12.31. Found: C, 52.32 H, 4.60 N, 4.94; S,12.27. Method 3 Benzylpenicillin phthalide ester:

The potassium salt of benzylpenicillin (20.0 g;10.054 mole) was dissovedin dry dimethylformamide (50 ml) and cooled to 0C. To this stirredsolution 3- bromophthalide (l 1.5 g; 0.054 mole) in drydimethylformamide (20 ml) was added in one portion. The reaction mixturewas allowed to warm to room temperature and then stirred for a further 2hours. The mixture was then poured into ice cold water (600 ml) andstirred vigorously. The white solid precipitate which separated wascollected and washed well with water. After drying, the material wasrecrystallised from hot isopropyl alcohol to give a white crystallineproduct (10.5 g. 41.9 percent). m.p. 167-169. The [.R. spectrum '(Nujol)contains inter alia strong bands at: 1770 cm"; 1678 cm"; 1524 cm" 970.n.m.r [(CD SO/D O] contains peaks at: 8 7.88 (4H.m. phthalidearomatics); 8 7.61 (1H.s. C0.0 CH-); 8 7.28 (5H.s. aromatics); 8 5.55(2H.m. B-lactams); 8 4.55 (1H.s.C proton); 8 3.56 (2H.s. PhCH CO)- 81.53 (6H.d. gem-dimethyls).

The purity as assessed by' hydroxylamine .assay was 109.2 percent. FoundC, 61.55; H, 4.90; N, 5.87; S, 6:72; C H N S0 requires: C, 61.80; H,4.72; N, 6.02; S, 6.86.

Benzylpenicillin phthalide ester (11.6 g; 0025M) was dissolved in drymethylene dichloride (250 ml) and chilled to 25. N-methyl morpholine(5.60 ml; 0.025M)was added followed by a solution of phosphoruspentachlorde (6.0 g) in methylene dichloride (150 ml) over 5 minutes. Apale yellow colour developed and after'stirring forone-half hour thetemperaturre rose to 0. The reaction mixture was recooled to 25 andN-methylmorpholine (5.60 ml) and dry methanol was added to give a slowsteady rise in temperature to After stirring at 5 to 0 for a further 2hours water (400 ml) was added with vigorous stirring whilst the pH ofthe mixture was adjusted from 1.2 to 6.0 with dilute sodium hydroxidesolution. i

The organic phase was separated washed with'water and saturated brineand filtered through siliconised paper.

A solution of p-toluenesulphonic acid monohydrate (4.75 g 0.025M) inacetone ml) was added with stirring to the organic layer and ether addeduntil the solution appeared cloudy.-On standing overnight at 0, 7.0 g ofwhite crystalline 6-aminopenicillanic acid phthalide ester,p-toluenesulphonate was obtained and a further crop of 2.5 g obtained byconcentration of the filtrate. Total yield 9.5 g 73.4 percent. n.m.r[(CD SO] 8 7.84(4H.s. phthalide aromatics). 8 7.58 (1H.s. -C0.0 CH) 87.30 (4H.s. sulphonate aromatics) 8: 5.50 (1H.d. B-lactam, J 4H2) 8 5.14(1H.d. B lactam. J 4H2) 8 4.68 (1H.s. C proton) 8 2.27 (3H.s. CH -)81.53 (6H.d. gem di-CH C H N S O requires: C, 53.08; N, 4.61; N, 5.39; S,

12.31. Found: C, 52.50; H, 4.62; N, 4.98; S, 12.34.

EXAMPLE 4 Ampicillin phthalide ester (1) via coupling of phthalide6-aminopenicillanate with an enamine protected A- aminophenylacetio acidmixed anhydride.-

Phthalide 6-aminopenicillanate p-toluenesulphonate (10.4 g) wassuspended in ethyl acetate (60 ml) and stirred vigorously with lN-sodiumbicarbonate (135 ml) for 20 min. at ambient temperatures. The organiclayer was separated, washed with water (100 ml) containing 2 percentsodium bicarbonate (5 ml) and dried over anhydrous magnesium sulphate.filtered and kept at 15C. 1

A mixed anhydride of sodium D(-)N-(1-methoxycarbonylpropen-Z-yl)-a-aminophenylacetate (5.4 g) in ethylacetate (30 ml) wasprepared by the addition of ethyl chloroformate (2ml) and pyridine (2 drops) at 15 and stirring the reaction mixture formin. at to C. To this mixed anhydride solution was added the ethylacetate solution of phthalide 6-aminopenicillanat'e and the mixedstirred at l5 for 15 mins. and then for a further min. without furthercooling.

Water (75 ml) was added, followed by 2N- hydrochloric acid (10 ml) andthe reaction mixture stirred vigorously for 25 mins. Petroleum ether,b.p. 60-80(25O ml) was added slowly with stirring. The aqueous layer wasseparated and saturated with sodium chloride and the oil which separatedwas extracted into ethyl acetate (2 X 100 ml) and dried over anhydrousmagnesium sulphate.

After filtration, the solution was concentrated in vacuo to ca onefourth volume, dry ether (ca. 250 ml) was added slowly and theampicillin phthalide which precipitated as a white amorphoushydrochloride salt (4.0 g; 40 percent) was collected and washed wellwith ether. Hydroxylamine assay 76.1 percent lodometric assay 77.5percent chlorine content 7.07 percent (theoretical 6.85 percent).

EXAMPLE 5 I 6-Aminopenicillanic acid (18.5 g- 0.085 mole) and sodiumbicarbonate (21g; O.25'mole) were dissolved in 200 ml of water and 100ml of acetone. To this solution, chilled in ice, was addeda-azidophenylacetyl chloride (16.6 g; 0.085 mole) diluted with 10 ml ofdry acetone. The temperature was held at 0 to 5C and the reactionmixture stirred for 2.5 hours.

The pH of the mixture was adjusted to 7.5 by adding a saturated sodiumbicarbonate solution. After being washed twice with diethyl ether, thereaction solution was acidified to pH 2 dilute HCl, and extracted withether. The ether solution containing the free penicillin was washedtwice with water and then extracted with ml of N potassium bicarbonatesolution. After 7 parent penicillanic acid present in the reaction mix-12 freeze drying the potassium salt of a-azidobenzyl penicillin wasobtained as a white powder (29.44 g; 84 percent yield).

The potassium salt of a-azidobenzyl penicillin (21.53 g; 0.05 mole) wasdispersed "in methylene dichloride (250 ml) and acetone (100 ml) and themixture chilled to 5C. To the stirred suspension ethyl chloroformate(5.13 g- 0.048 mole) was added dropwise followed by a catalytic quantityof pyridine. The mixture was stirred at -5C for 30 minutes. vO-phthalaldehydic acid (6.5 g; 0.05 mole) was then added to the reactionmixture and after a few minutes,

Catalytic hydrogentation of the crude phthalide ester ofa-azidobenzylpenicillin gave the phthalide ester ofoz-aminobenzylpenicillin' which, after purification by chromatographywas found to be identical with an authentic sample prepared by themethod ofvExample 2.

EXAMPLE 6 The rates of hydrolysis of the phthalide ester of 6- D()a-aminophenylacetamido] penicillanic acid HCl were determined byincubating the ester at the equivalent ovSag/ml6-[D(-)a-aminophenylacetamido] penicillanic acid in /20 pH 7.0potassium phosphate buffer, percent human blood and 90 squirrel monkeyblood. The ester was also tested for hydroxysis in squirrel monkey smallintestine homogenate at the equivalent of p-g/ml 6-[D(-)aaminophenylacetamido] penicillanic acid, the reaction mixtures beingdiluted to the equivalent of 5.0 ug/ml of the parent penicillanic acidbefore assay. The tissue homogenate was prepared by homogenising washedsquirrel monkey small intes-v tine in four times its weight of "/20potassium phosphate buffer. This stock preparation was diluted a further1:10 for reaction mixture.

All reaction mixtures were incubated at 37C. After incubation, the esterwas separated from the reaction mixture by electrophoresis. 5 ,ul.aliquots of the reaction mixtures, and 6-[D(-) a-aminophenylacetamido]penicillanic acid standards prepared in the appropriate medium, wereapplied to starch-agar gel plates buffered at pH 5.5. A potential of 15volts/cm was applied across the platefor 20 minutes. At this pH theparent penicillanic acid remains near the origin and any ester presentmigrates towards the cathode. The amount of tions calculated.

- RESULTS Hydrolysis at 37C to 6-[D(-)a-aminophenylacetamidolPenicillanic acid at:

Hydrolysis M i m 3 mins 8 mins 15 mins 25 mins Acid (pH 2.0) 0 0 0 0Aqueous Buffer (pH 7.0) 10 15 20 25 Human Blood (PH 7.0) 50' 62 80 v 84RESU LTS -Continued 70 Hydrolysis at 37C to6-[D(-)a-aminophenylacetamidol Penicillanic acid at:

aminophenylacetamido] penicillanic acid of formula (I) or apharmaceutically acceptable acid addition salt thereof:

2. The hydrochloride acid addition salt of the penicillin phthalideester of claim 1.

e Page 1 of 6 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OFCORRECTIQ PATENT N0. 3,860,579 9 DATED 1 January 14, 1975 |NV ENTOR(S)Harry Ferres and John Peter Clayton it is certified that error appearsin the above-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 1, line 2 'pathalide" should read --phthalideline 41 delete "to".

Column 2, line 13 "commited" should read "converted- Formula (III)should appear as R R i q K (In) line 44 "X" should be -Z--.- a Column 3,line 47 "phthalido" should read --phthalide--.

Column 4, line 59 v1 should read "(VIA)".

Column 5, line 5 "(VI)" should read (VIA)-. at line 27 "(v)" should read--(VA)--.

Page 2 of 6 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OFCORRECTION PATENT NO. 3,860,579 DATED January 14, 1975 INVENTOMS) HarryFerres and John Peter Clayton It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 5, line 28 "(V)" should read --(VIA)--.

line 51 "convention" should read "conventional".

Column 6, line 6 "Nbromosuccini" should read --N-bromosuccini-- line 23"(17.5;" should read --(17.5 g;--.

line 43 "nbuta" should read --n-buta--.

Column 7 Formula (I) should read as s CH3 Br 6 CH l d/N.\-l 0 60 K CH it5 G Page 3 of 6 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFI PATENTNO. 3,860,579

DATED January 1 |N\/ ENTOR(S) Harry Fer GATE OF CORRECTION res andJohn'Peter Clayton It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 7, line 32 line 64 Column 8, line 11 line 37 Column 9, line 16line Column 10, line 66 line 67 "tectd" should read --tected--.

"portion" should read --portions-=.

'Yl" should read --yl--.

"declourising" should read -decolourising --aminophenylacetic--.

Page 4 of 6 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OFCORRECTION PATENT N0. 1 3,860,579 DATED January 14, 1975 INVENTOMS)Harry Ferres and John Peter Clayton It is certified that error appearsin the above-identified patent and that said LettersPatent are herebycorrected as shown below:

Column 11 The formula should read C'IH-CO N D(|3H-OOOC0OC H /N CH C H CH--C H 3 u E H I CH CH 0 \C)J \C/ l 6APA phthalide OCH OCH (formula to becontinued on next page) Page 5 of 6 UNITED STATES PATENT AND TRADEMARKOFFICE CERTIFICATE OF CORRECTIN PATENT N0. 1 3, DATED January 14, 1975-mm Harry Ferres and John Peter Clayton It is certified that errorappears in the above-identified patent and that said Letters Patent arehereby corrected as shown below:

Column 11 (continued) Q Column 11, line 35 "mixed" should be-miXture--.,

line 66 After "pH 2" insert --with-.

Column 12, line 20 "hydrogentation" should read --hydrogenation-.

4 a a Page; 6 bf 6 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATEOF CORRECTION PATENT NO. 3,860,579 A DATED January 14, 1975 i yE T0R(5)Harry Ferres and John Peter Clayton It is certified that error appearsin the above-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 12, line 33 "hydroxysis" should read --==-hydro1ysis--,

a Signed and Seated this Tenth Day of August 1976 [SEAL] Arrest: t

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner ojPalemsand Trademarks UNITED STATES PATENT AND TRADEMARK OFFICE CertificatePatent No. 3,860,57 9 Patented January 14, 1975 Harry Ferres and JohnPeter Clayton Application having been made by Harry Ferres and JohnPeter Clayton, the inventors named in the patent above identified, andBeeoham Group Limited, Brentford, Middlesex, England, the assignee, forthe issuance of a certifioate under the provisions of Title 35, Section256, of the United States Code, deleting the name of John Peter Claytonas a joint inventor, and a showing and proof of facts satisfying therequirements of the said section having been submitted, it is this 6thday of April 1976, certified that the name of the said John PeterClayton is hereby deleted to the said patent as a joint inventor withthe said Harry Ferres.

FRED W. SI-IERLING, Associate Solicitor.

2. The hydrochloride acid addition salt of the penicillin phthalideester of claim 1.